HU230404B1 - Dilution based inhibition assay - Google Patents

Abstract

The present invention provides for a process to assess the level of reversible inhibition of an enzyme by an inhibitor, in particular in the field of assessment the effectiveness of a medical treatment. In a particularly preferred embodiment the effectiveness of angiotensin converting enzyme (ACE) inhibitor (ACEi) therapy is disclosed. The invention also relates to uses of enzyme substrates and kits for the assessment of inhibition level as well as an apparatus designed for use in a process of the present invention.

Description

FIELD OF THE INVENTION

The present invention relates to a method for determining the reversible inhibitory effect of an enzyme inhibitor, in particular. medication: treatment to determine the Immunity. According to a particularly preferred embodiment, the efficacy of a blood tom scavenging drug was investigated. Particularly preferably, the method is? The present invention relates to: enzyme s. uM / tms, xt η η n (the use of kits for determining inhibition as well as the use of kits). a kw. mes, rueL in the procedure described in the mteitans ban

TECHNICAL POSITION

The problem of determining the age or efficacy of anti-enzyme inhibitors has openly been raised. In these methods, it is first of all necessary to design an experimental set-up, typically comparing the enzyme activity of the treated patient to a "normal" activity symbase of a healthy control group or a control group in which a. treatment is effective. However, this traditional method: raises many problems. For example, there is a need for a control group in which there are different parophinology features (for example, they do not suffer from the same disease): and individual differences mn; nílusinak.

Patients' attitudes are another problem that often appears on Lem 1 Knk. when we need to collect samples many times to cover the effects of a treatment. In spite of all these nannies, many patients refuse to visit their physician on a regular basis, which makes the assessment of their strangers possible.

Moreover, direct activity measurement is influenced by individual characteristics, such as genetic variability. However, this does not indicate the efficacy of the inhibitor: it is necessarily related to it (Figure 8).

Another method by which the efficiency of an enzyme treatment has been achieved is to determine the presence of an inhibitor in a given or obtained body fluid. However, while this series of experiments tracks the drug's blood rate, it is not able to track the actual effect of the drug on the body. A further problem is the preliminary purification of the inhibitory effect of the inhibitor on the enzyme and the analytical methods used are time consuming and expensive.

We may face similar problems, measuring the stkatjitk of arsukss · an impersonal abomination. Many attempts have been made to solve these, and similar ptobíemes can be found.

Publication number HS21140408A957A1 measured the amount of HVV protease inmbhor in a conjugate complex. The method was based on the wheezing of the conjugated inhibitor and the inhibitor to be measured.

In the WÜ2O01085985AI, the overlapping transducers of synstrate-specific fluxes were found to be useful, however, many of these samples were obtained from several patients at different times. reagent diluent was used, added to it with a potentially defective method and an efficient one that was linked to the enzyme as a cofactor to cover the genetic error,

In WO2505255601A1, the plasma-to-plasma cone ratio and the ratio of plasma-to-peak volume ratios were plotted against different dilutions in blood samples over time.

1CVO0L142ISG, however, is quite assembled and bed-like that you are not studying a speet-containing enzyme-inhibitor pair,

The PCI u 820814) 45643 arable-ash free-standing planter®. you have written a correlation between the ACE inhibitors and the degree of inhibition and the occurrence of ttz, angmoedema, Ex method differs from the single-dilution overlay used according to tslncr.

No. 88272: 8285 was reported to detect the presence of mmbiferOk by means of a yeast peroxidase activity. This method is unrelated to the ictea invention

K .. Edited by Sandy Pang, A. öavtd Rodrignes and M. Rtóxaund M. Peter, "Enzyree attd iransporter" -no dne ig nte M ons víu kon \ «nncet \ ude Neu V t. 2 ^Λ 1ό 14u \ v ”8 '44 o igxo ₅ Page 188) the method for characterizing the enzyme-inhibitory relationship, batting the -.mhhich constant determination? describe. however, the mhtbuors added to the aftermarket P4S0. were given and indicated their attachment. The method was not used to determine the level of inhibitor or to measure the effect of inhibition on -5 y cells.

The traditional dilution-based etregregulation inegulation? In the case of enabling methods, only the concentration of the inhibitor is changed, the conversion of both inhibitor and enzyme to the method known in the art is diluted.

So: there is still a need for a fast and reliable ® method to reverse the degree of reversible inhibition of an enzyme,

SHORT DESCRIPTION OF lALAi.MANY

Slime is a method for re-maintaining the degree of inhibition of reversible inhibition by an enzyme inhibitor, which process itself is brick ^! and the following:

(a): assurances of at least one starting sample (hl excluding at least seven subroutines from said at least one starting sample;

(e) preparing reaction mixtures containing the various dilutions of the aliquots and, if desired, these aliquots; keep dilute creatures diluted with toxins! orozns, thus ensuring that the active and inhibitory forms of the enzyme are present in varying proportions in these reaction mixtures when the inhibitor is down;

(cl) measuring the ethnithelitic enzyme in the digestion mixtures to obtain a measured yeast cooling value;

(multiplying the measured acute values by the appropriate partial factors to determine the calculated etymic activity values;

(1) Determining the extent of reversible en2smg inhibition in at least one initial Climate, where: the calculated edema activation values are ^e S g g (correlate with >reverse inhibitory ».

Preferably, the enzyme is present in the reaction mixture. In a preferred embodiment, the enzyme is present in at least one of the starting samples mentioned. In another preferred embodiment, the enzyme. an enzyme isolated according to a preferred embodiment, or more preferably a purifier, as an additional ingredient to any of the samples, preferably to said reaction mixture? or recombinant enzyme.

In a preferred embodiment, said at least one starting sample is a single sample. Another advantageous embodiment is the snodokhah of the embodiment - said: at least one degree sample of a single hot one, for example; from a patient: either from a blend of horseradish or from an artificial blend: comes from an example! a mixture of heads formed from an industrial process or aortic aorta, Sakeeoer discovers that a knife or multiple starting samples may be taken from the same spelling, preferably at a single time :. Preferably, two or more starting samples

18882-4423 / SG is obtained either as a sham, which consists of the same or essentially ngyaoazon constituents.

In a preferred embodiment, the at least one starting sample is a biological sample. derived from a biological organism; Mirto-in. or a biology! from Niintabo. In a particularly preferred oregativity subtype, at least one efflux sample is or is derived from blood. Preferably, the biological: sample obtainable living organisms This living animal is preferably an animal, more preferably a vertebrate, more preferably a mammal, very preferably a human.

In another embodiment, said at least one snatch; mima micro-organisms. emanating from its release. Microorganisms are understood to include any prokaryotic or encephalic cell killer.

Preferably, the at least one starting point; samples from a "process" that is stolen from said Iabibitor ttsc ₍ ís'gvk e e'tte <íc ^! a \ zna ne \

Preferably, the method is performed to determine the efficacy of drug therapy.

A very preferred embodiment of the invention is when the enzyme is the angiotensin-converting enzyme (ACF). and an inhibitor of said inhibitor ACE (ACE1).

Forewarning: the cooling factors are set in bed. that they are statistically. significant differences in addresseeerye / reí. a ^ untfoft c'v mrkíitü also in etiquette, Jemotoa iegaí.é'ő sauy o ereoseoKm.

for enzymatic activity at least ammunition or higher, when calculating the calculated azimuth values at high dilution factors with the calculated e. e. at the dilution factor obtained with a low dilution factor. iabibitor is present in said reaction mixture. Preferably, said dilution factors are potentiated based on a predetermined concentration of inhibitor. in the case of at least one starting sample and based on a predetermined dose effect of the inhibitor and the enzyme, preferably the hormone factors are calculated. parameters already known or previously determined, such as: inhibitory dissociation or inhibition! or the rate constant of the reaction. In a further preferred embodiment, the dilution wood throat. experimentally, by describing &quot; in the invention; procedure, different dilution! alchemists. Preferably the dilution; factors are determined before a start; reversible inhibition of the sample; its level is estimated in accordance with the invention,

A further object of the present invention is the use of scavenges of an enzyme, and optionally further zagems, in order to allow the immortalization of reversible inhibition of zeeze in said tendons.

A. The invention also relates to the use of an isolated enzyme in the method of the present invention. the degree of reversible inhibition of the enzyme by one. The preferred enzyme is purified or recombinant. Very preferably, additional reagents other than the substituents or more; or additionally used to measure enzyme activity in the method of the invention.

The present invention relates to a device for determining the degree of reversible inhibition of an enzyme (instrument) by an inhibitor &lt; / RTI &gt;

- means for juggling or creating at least two abqnotes from the at least one starting pattern;

tool to further creator; added north to the ahguots: creating reaction mixtures in forests »;:

means for quenching the reaction mixtures at various dilutions;

- means for reacting said reaction mixture; arc to measure the activity of the enzyme. measures. activity to gain values;

109902-1421 / 40

- a means for determining the extent of reversible inhibition in at least one cleavage radar by assisting with the values of the measured activity: with appropriate clamping patterns to obtain calculated euzinr activities. In doing so, the difference between the calculated enzyme activities is proportional to the degree of reversible inhibitory cleavage.

The present invention relates to the use of kits according to the invention for s στ »e ^! · A'-ban the reversible inhibition of enzyme for the determination of blistering kits containing:

- the handwheels are stored in the description

- substitution of the enzyme

- optionally, said enzyme in an isolated horseradish

- optionally, an inhibitor of said enzyme

optionally a dilution buffer

OkfeNíClÓK

The singular plural in the description, "one," and contextually, the "you," plural include the plural, unless? .vrb ours what e / J who aunt / price;?,.

A. The word "isolated" means "human hand is alpha! medosro from the natural state. If a ravlekttla or substance occurs in nature, then it is "taste" if it is a tenderness and 'e.

The word "includes" or "includes" or "contains"; what I mean in the description is that this; the meaning of the words nesn is complete, that is, chimerites are, in addition to the properties, methodological steps, or components listed above, breathe in additional properties, methodological steps or components.

The expression "essentially consisting of or consisting of" means those properties, either methodological steps or components are essential, but also additional fluid or methodological steps or components which are not significantly affected by the application, process, composition, or other important attributes are a basic sign of 'F'etVos: understand that in this case,' includes 'or' in the dude 'or' \ ates; bo! standing expressions may be replaced by "consisting essentially of" or "substantially containing" the term, without further mention of facts,

The terms “other sources” or that “samples from a single source” refer to a bed hrsgy existed, or there was a mixture from which the samples originated / were derived from, or were spammed from said mixture : like they were made from or oyor, oh, um <c «at the same time. Preferably, the composition of these samples from a single source is the same in lemon skies.

The term "substantially common" bed is understood to mean that it is sufficiently close in time to ensure that the composition of the unique source did not substantially change, so it was substantially the same during sampling.

The expression that two samples are "essentially the same composition" (for example, when samples of the same composition have been taken at different times) means that the composition of the samples is sufficiently similar to »produce reliable results from the two mimes.

109 903 - 423éSG !.

V \ 3R \ kB>) vU) Fig.VA t - Dose effect of Csptoprti on the activity of the brown fungi converting enzyme

Ascending Captuprius urinary tract: non-linear inhibition of human serum ACE activity. Therapy for the inhibition of wxis (as with ACE inhibitors) is: inhibition of the enzyme as much as possible. Dosage of the enzyme medications: however, there is a limit to the increase in their activity. Therefore, the inhibitor xmoenavir used in clinical practice. in this case, it is ACE inhibitory), which is mostly in the Sahniacinic dessert range (shown in the figure as a temporary effect). It is characteristic of the region that small changes in the concentration of the thymus can lead to large differences in the actual inhibitory effect. decreases to a concentration of csptopril of 30 tiM salts to 3 ttM (shoot dilution),

Figure 2 - Effect of Dilution on Inhibited Rehabilitation .ACE Activity

Recombination was added to ACE: ACE inhibitor captured: (8.1, id and 100 nM, bumps are depicted in the figure}, maid ACE activity was measured & diluted (1x) and diluted (max. dilution had only a minor effect on zW £ activity when the initial inhibitory effect was low (eg 8.1 nM, In contrast: significant increase in ÁCS activity was detected.): dilution resulted in a relevant capiopril concentration. by: (shoot nMk Finally, at higher concentrations of eaptopnl (100 sM), we only saw a significant increase in ACE activity at na g \ eb? t eg: b- »zores),

Figure 3 - Theoretical effect of myocardial blockade on human ACE inhibited by eapiproprid

Equation 1 represents the general model of reversible enzyme inhibition, where the actual enzyme activity in the reaction mixture is dependent on the hunt iv dictation. Enzyme expression values were plotted at i-512-fold dilutions of the O-iböb nXEcapiop ti series. assuming nations (denoted as different symbols in the figure!), we estimated the 2 "serum \ CE concentration based on the 10 awt endeheheh data, Matching the dots equation 1, amur mrtest The figure shows that the increase in enzyme activity from the dilution factor ( depends on the predetermined (or known) properties of the enzymehtbstor interaction (e.g., Ki, co-operative factor). In the case of inhibition, the inhibitory effect can be estimated by comparing the low (in this case the enzyme is irklmor bound) and high is: enzyme activity values measured for their most active, active state (vsa) dilution factor. The degree of inhibition can be determined when it is low. the phosphorus enzyme activity is compared to the high dilution case moghstart tens,

Figure 4 - Calculated serum ACE inhibition in hypertensive patients

The applicability of the inventive method of smoking is demonstrated in the treatment of the agglutinative osmosis enzyme and in patients treated with an inhibitor of this enzyme. Serum ACE activity was measured in vein samples. Hypertensive patients were included. Some patients received: ACE-gel · (marked as + ..ACE-blocker), while others: this drug was not administered (marked as control). A s: -cr ' ^! -m-mn ^! The extent of ACE inhibition is measured by measuring the levels of each patient with ACE inhibitor therapy; It is clear that some patients in the ACE-inhibitor-treated group had similar ACE activity values: thin, mim in the untreated, control group, but it is also obvious that in the majority of patients with hands »alesunyahbh enzyme file · values were measurable.

Figure 5 - Different degrees of serum ACE inhibition in intestinal tract

When the extent of ACE inhibition in patients was estimated by the lein method, a surprising fact emerges from the i89k02-i423 / SC. In untreated patients who are not taking ACE inhibitors, significant <RTI ID = 0.0> (7S%) </RTI> ACE g.r av • 'can be elerstg <g> cndorm \ Cíga So efen »etec A't \ fesfes. \\ v AhE gafk-vJ ss \ el.

Patients typically had much higher ACE inhibition (Data are shown in this section). The line represents a selected hypothetical value (UHF, above which you determine with the Vtl · j, at'o temp 'on the head, oh, - Dilution® »method: the degree of ACE inhibition is proportional to the clinical target of the treatment.

Estimates of ACE inhibition were performed as described herein. The ACE activity of 4x and A0 diluted carbon samples was determined and the blood pressure of the patients was measured. The extent of apparent ACE inhibition was calculated as the quotient of the values of 4-5 times and 4 times the activity. Patients have mean arterial blood pressure values below 9tEb in the figure. Above s _: (sr3 18} for ACE inhibition. Columns represent mean values and stemler error, asterisks indicate significant difference between the two groups (p “to, Ö93), Insufficient. we have shown that the present invention is useful in the treatment of an enzyme inhibition with a Reversible Inhibitor-directed treatment: for the treatment of clinical Itafesoniosis,

Figure 7: Elevation of l'vopenic AC1> activity by dilution of ts serum

The process described in the invention suggests that the required dilution! factor may be determined by taking into account the Ki value previously determined and the dose-response relationship of the inhibitor to the enzyme under investigation. Exogenous ACE activity was measured to determine the extent of ACE inhibition in the serum secretion of a patient taking ACE inhibitor. Three measurement results; illustrated in Figure (mean AKD). The results: show that inhibited ACE activity is significantly increased by dilution. Especially when you consider that the ACE activity of the non-diluted myrrh was increased from 1 AV m to more than 1 IM ACCml for High Dilution Drink (128-fold).

Figure 8 - Staining Concepts for Determination of Nz Enzyme Activity

The determination of the efficacy of an enzyme-inhibiting therapy in the conventional manner is based on comparing the enzyme acoustic activity of a treated group with an expected inhibition) and a control group with no inhibition expected. a reference range is determined from the values of the control group: enzyme activity, and the cymyme activities on this. reference tarícntámiya! let's compare. Values for untreated patients, however, are Ί> κfe'z K-uc \ for the given inverter from sertroctol \ alanon; as' as erztn .Atsssfsvi eeta'so / l: t s txtcg »eg progresses. In this study, we compared the treatment of two groups of hypertensive patients, where the control group included patients who did not receive an ACE blocker (ISA patient), whereas patients who received an ACE blocker included 1.587 patients. . It is striking that a significant (about fourfold) varicose vein has iron in the ACE activity values of the untreated, ketbilöll group, with a 20% overlap between the two groups. This makes the decision that a. the patient has relatively high ACE activity with iron successful A.CE inhibitor therapy or the patient has insufficient ACE inhibitor therapy,

Figure 9 - Improving the clinical efficacy of the described invention

Patients whose ACE inhibition was less than 90% as determined by a single sample using the method described herein were contacted. The patients concerned were asked to devote my due attention to their medication, with particular reference to: ACE

IV04o2-! 4 25 Kfe inhibitor for proper use and icex for further examination. In some cases ». the. patients, inhibition of the enzyme was greater than 90% by nmodA MAgaU. There was a strong correlation between the blood pressure of the patients and the medication catheter bfetesAg wghatarotM. efficiency. It is of particular interest that patients have a.i.wnyubb blood pressure when it is: the degree of GABA inhibition is greater than a. 99% versus 90% aUtd inhibition. Mean values are shown in the figure (<path * S £ M),

Figure 19 - Narration between ACE Messiness and Activity

Lung ACE was measured by ELEsA, while activity was measured by EAPGG hydrolysis atop in the same sample. Patient genotype was also detected in patients with pathology, DD genotype had 58% higher serum ACE levels than gemtype II, activity was significantly less understood by the genotype.

you. Figure ~ Indications of endogenous bhilblter

The inconsistency observed between the AGI tendon and the afferent may be due to the presence of low ACE activity mutants. mw is not a suitable method either. For the determination of ACE activity, an artificial striatum is defined as the substrate for the treatment of ACE with Salamia angioedema; ítasznáityk. Close Autumn SfiCgert See Me ». in kCl activity along with the lymphatic degradation of these substrates, which is supported by the aptitude assays for EAPo. A. In the following, the effect of dilution on enzyme activity was investigated. The Teruo ACE activity was not linear, but an increase in the dilution degree of increase, increase which was observed rekotnbsuáas, upon dilution without occurring serum purified ethene components This observation suggests a any allowance ishihíter presence of _{sz.étmsbas,:} described in accordance with the invention · claims.

Figure 12 - Molecular weight of the endogenous barrier cyst of myocyte ACE

Serum samples from healthy untreated volunteers were screened through hairs that were transient to S9 kDa or 100 kI saturated molecular weight proteins. Filtration with a 59kEto filter had no effect. nn'g filtration with a 100 kDa filter significantly altered the changes in dilution enzyme activity. It is noteworthy that significantly higher activity was present in the filtered mimics-u · I pl (10 ki ki.ia-o $ filtration s.

Figure 13 - Inhibition of serum ACE in myo alfeussteal

Haemorrhages to identify more than 50 kEtenal but less than 19 kDa of endogenous Inhibitors have demonstrated a link between serum ACE and hay alhmnin. The effect of serum albumin on recombinant ACE is first investigated. In increasing sulfur catheterization, the teacher inhibited serum albumin by vapor concentration, recombinant ACE activity, and a Ki value of 9.6 _: mg fml. Considering that the albam strain has a range of 49 to 99 mg / ml, the physiological serum ACE gasification may be due to the presence of hairy almond. To counteract this hypothesis, ACE was purified by human body onM e »<zen test subjects inhibited the effect of human stream albem on the Hwua serum dream in a similar manner to the lysitic iCi. akuvlkísá, tatai as he did with the recembinance ACE. Finally, your claim of the invention to detect the presence of an inhibitor by exegase addition of the enzyme to the raaxial mixture was tested. Serial dilutions: prepared from a sample containing recombinant ACE and potentiated serum aibmnin. A large increase in the calculated ACE activity can be experienced »already at a slight dilution in the fpi range. 2-fold and 8-fold bigriassoai.

199992-1423 / SG

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a quick and easy way to suppress the magic of an enzyme inhibitor system. k «from the sample. This method in Serbian does not require the purchase of multiple dead or snacking mhtívaf for sneg

Between the enzyme and the idiots! jig-effect combineShuffle with a sigmoly steamer! as shown in Figure 1. based on our own measurements. In the figure, the most important phases of this inhibition are the following, of which the following are noteworthy: the slightest aspect of the inhibition under maximum saturation conditions Each inhibitor molecule binds to each enzyme molecule); transient inhibitory hernia can be observed at lower inbibiter concentrations; nng «binder becomes ineffective even at lower concentrations, free of blinding effects, maximum enzyme activity). The clinical effect of inhibitors used as a drug is usually transient so that the inhibitory effect is significant but not attained. x '0' hatching.

Theoretical observation is that the stability of any reversible i mmn ^ r-enzyme complex can be characterized by a constant (KI) and the free enzyme (E), the free mhibite? »Π v- enzyme inhibitor complex (E1) costration by the following xzerym: calculate {!. equation):

Κί-α'ΊίΕΙ (!)

The ratio of the concentration of the inhibited enzyme is see »» _v n. lm concentration, which is the path to the degree of inhibition of acta (Inh) according to the following equation:

Ιοί) - ^: · Η <r ti) (and

The basis of the present invention is that the minds of the nerves can determine the maximal enzyme activity of the mima sufficiently, with high excretion. Under these conditions (four dilutions), the enzyme-monomer (E1) complex may be machine negligible relative to the free enzyme coscentra (E1). As an example, consider a reversible enzyme inhibitor: if the sample is measured by brain enzyme inactivity on at least two dilutions, the most severe sample gives the activity of the inhibited enzyme (Einh). The activity of the higher dilution-measured sample tends to be that of the activity measured under conditions such as that of the inhibitor d-xxxom.fi in the eo / norol czalial life of the enzyme akin belly) xz mhfoitornemet · conditions measured xm. (similar to when the patient does not take the prescribed medication!). This requires: met.vckcnl: the maximum enzyme activity of the sample (Etnax when E '-'-Ei) can be estimated between the diluted coru.met.vek ^ _{} 5} ^ _{! 5} ek basedm,. 'Divine, kenshncnx onto co.vat a / et ο nm ό u-sv ti os, o- ^ \ .ntt rn íbun is). K. and the actual degree of inhibition of enzyme in our cell El with a significant concentration of iron Jetem) is the least diluted: it can be estimated from the sample (under these conditions most of the enzyme is in inhibitory bound, inhibited form).

The degree of reversible enzyme inhibition can be estimated (b) within a single barn using the following equation (T equation):

inh Einh En-ax {fo

The present invention provides an alternative method for determining the degree of reversible enzyme inhibition in experimental samples. It is a great advantage of the invention that the direct and reversible eosymphography of a single sample can be measured without the need to compare the kap-vt credential with the results of any other sample. Direct determination of the extent of reversible enzyme inhibition may be desirable if there is no appropriate control or if a particular sample is to be inhibited accurately! value. These applications may have included the control of the effectiveness of medication to inhibit an eazlm,

1-1993) 2-1423 (Sö fö -> \, o> s _V ipi _v n i.ek vi ilaj-nakoiog okig,>? T> 0 «• u / unek Vikinek ntríearck pl ang.etén. M keoscitta enzyme, retn® H1-V proteases, melting order subunits: salt® fistodesterase-S (e.g., β-dbdiosterose-S, etc.), reductases (e.g., dichydro-bile reouctase, Bhd0-CoA redbase, soda, ct-oxooxidase, moooamib. The method can be used to detect the presence of toxic substances from living organisms or from fluids in the circulatory system. In the broad sense, the method described is applicable to a pair of membrane enzyme inhibitors, for example, but not limited to the following enzymes;

Aideld-dehydrogenase, Motmaminoxidase, Cyclooxygenases (COXk K-Vltamy epex redox, Aromal® luamsterol denrectase, Uproxygenases, Thyreoperomdase, Axlotsrool-γ-δ-jdidase, Inoshrraeafö). , Diphadrophotofluidine kinase Spuakmeredrinase, 14-reductase, Xanthine oxidase, 4-Mdfxifomylpinivate diexigenase, R5Oucleosyldisphosphine reductase, Protein. DNA polimert®, Csaba trarazararaáz, tyrosine kinase-öÍieinanmd íiboH «kk; eiid-fernbb tmoszteráz, phosphoenolpyruvate-tmöszfortLZ brown] eüoszólüeas elágazottláacö ammoiransxföíáz (kBCAE :) Astyartíl-prozeázok, Reston Asgioteazsö converting this butt, Dlpeptidil pepitátok, Carbőxipepliíföz A, Human encephalitis, 2S proiessema, Eostbase yeasts, Ghcosidazofo Lipases, Cakmeurin, exterminated by polysilicon AAS, .TOFA decarbosylase, Carboxylic acid aldase, Rismid docazboxylase, Oraithithio decarbaxase, Aiasis racemase, DNA glycases, Buccal meseasases, b, 7-®oerase, Dlhydropteorase enzyme, Timlalease simase, Glucosylase base, Glycosyleramide silylase.

In that case, the primary areas of the invention are pepitates and proteases.

The present invention may be applicable to: Inhibitors which are used to delay enzyme-linked diseases or which have been identified as drug-loaded in such an enzyme. It is fibrous to note that it is described herein. The method can not only be used to measure the actual inhibition or activation of a biological target (eg, a deactivated target) but can also be used. 'mg' · Measure the inhibitory efficacy of an inhibitory agent in the blood for a cclpvnt cellular space (pb: dipeptilyl peptidase-4, tnertoamw-oxclase)

The above-described theory suggests that dilution can be used to determine the enteric inhibitory potencies of a reversible salt in a single salt, irrespective of the actual enzyme machinery tyrosine and the specific activity of the enzyme. , where the efficacy of angiotensin converting enzyme inhibitory therapy was investigated.

The aoglycid converting enzyme (ACE) is an α-metal metallo-endodipeptidase that catalyzes the conversion of snood photoinhibitory 1 to 11 carbon monoxide, and is taken up by other peptides in the metastasis, such as: Recení advanees in teosvkdge of tlte sbuctnrs and foaeion: of the angiosensin 1 com-erting euzyme I Hypertens Suppí 1995; LfoS3-10. C et al., 1 Rio Ebem 199 i; 266: 1537? Ss. The somatic form of ACE is introduced into the circulation by the ACE secretase (Öpporsg SY et al., Böchem 1 1993; 292 ('Pi 2)): 597-693.1. ACF is part of the remn-angiotetmin-aldospherea system ("reom-rntgioiensls-aldosförono systeirt'b RAAS}, an important regulator of blood pressure and salt-water household (PfoSérMAei ah, N Engf J Med 1992; 327. 669-77).

ACE-inhibiting drugs are one of the most popular and. the most commonly used therapeutic moddeters

199992- l-Efö.'SG

RA - \ St »ak. .Al E-gateways reduce the incidence of thousands of patients with pathophysiological cerebral palsy. bzasnos ianuhnany found that their ACE-barrier rainbows are more likely to improve kirtdiovascular mortality, the likelihood of fatal fatal myocardial infarction, or cardiac arrest in stable coronary artery disease (Pox O1 Chain 2003; 562: 782-S,), ö : written. IA® Col! Cardtol 1996: 27: 337-44.), reduce 5-week reportability after myocardial injection (1S1S-4 write, Chain 1995; 345: 669-35,), Reduce: Moralization of heart failure ICoba E4, et al., N Engi. 1, 1991: 325: 303-10.) And inhibited: left ventricular remodeling (Grgeaberg et al., Circdasmn '95, QI: 2573-81.). ACE inhibitors delay the onset of hypertension (Euders S, et al., J Hypertens 3008.26: 1-187-96) and reduce the left ventricular mass index value: left carotid hypertrophic tCuspldi C et al. .1 Hypeneas 2002: 20: 2293-300.). Reduce the ictia of ntikroalbujsiauna and the risk of developing transient nsfepatine in diabetic nidlitazyme 2 (Ruggeussb P, N Esgl 3 Me 2004: 351: 1041-51,) and further reduce the risk of new-onset diabetes mellitus, v í. n 'Mrd 2 Ct c ⁷ ' sots 1 _{/ s} ss í1 ub iatok 0) ΙΛ u, »>'<. The field of V / κ> .r o-> is suggested by the mention: diseases in the ictnc-nec-anise-nis. His studies have been carried out to investigate the link between ACE and sword saphenous disease. Art has been found to control the ACb expressor by genetic control of the ACE gene lymphoid and CDF polypeptide (Rigai B el al, 3 din Invest

1990 :: 86: 3343-6,), Increased ACE due to presence of AD allele was harmed as potential risk aviator tvui-vn i refers to Satui? Hundreds of WfoC'fo ³ n -'- tv tree fans have investigated the role of ACE gene typing in & nzxcubic assessment and optimization of ACL inhibitor therapy. These studies have killed the long-standing genotype of ACE in patients with the D1 genotype, with a concentration approximately one-and-a-half times higher (Riga B et al, .1 din cred. 1990: 86: 1343-6.). E. genotype), but no positive finding was found, ^ Zvlu „ _o v ^ a scium \ v 1 ixmixreg o \ mlotostodar ·» cet? wk gentlemen,> vK e ϊοα a? \ t 1 inhibitors have proven to be a successful therapeutic treatment tube.

These data suggested that the expression differences expressed by the genotype in the expression of U? CE can be well tolerated by individuals. This is further supported by the fact that the D allele occurs more frequently (509 ») (Lindpaininer K, et al. N Esgl. 1 Mied 1995: 332: 71) 6-1 !,) and ^: evrthiciósau sorah ballroom.

\ rtamk yt i / nit íogv <. ta j to the capvAÍa ο o \ Ci ge \ nm ~ - v \ p ese late action. Based on the method set forth in the claims of the present study, it has been found that ACE activity is regulated by an endogenous inhibitor that reduces ACE activity by at least 73% in humans by inducing ACE activity over a wide expression range.

PFIDAK

For a number of healthy volunteers, we added an increasing amount of APC inhibitor eapipropril and determined the activity Λ gates of the endogenous angioteturotrophosphatase enzyme of nbsis as shown in Figure 1. It is observed that the degree of inhibition is in the hands of mraomabic saturation conditions, while the inhibitor becomes ineffective at ever lower concentrations (variability, maximum enzyme activity). Drugs used in medicine usually lose their clinical effect during the transient phase of the curve, where target inhibition is significant, but less than complete inhibition.

The non-feasibility of the method was first tested by recombinant ACE-nilil which was mixed with an ACE inhibitor (eaptopril) and diluted with maid sm-vste-cm (Figure 2). That's: proof of the result. The dilution of the sample is due to a <RTI ID = 0.0> growth </RTI> of inhibitors (1) and the enzyme:

109902-1423 / S0 (E). dissociative, which results in inhibited AGE (E1). excess concentration tubes. it is. also: it was visible, leaves it. AGE. the increase in activity is proportional to the degree of inhibition: the nominal change in AGE activity is negligible when the initial barrier is negligible (eaptopnic concentration OJ okit, there is a significant increase in low dilution factors when the parent inhibitor coagulates with the parent Finally, the concentration of the inhibitor up to the point of snaxylation · barrier se se m * 1 A! ρ <. hujT v git „t J · o harm ix \ ^! k \> π t> i - tet lake \ h it. * · Y level activity should be released from inhibition (Figure 2).

Hereinafter, test the method. In the course of her life there was a series of Kim Kilkese investigations.

Estimation of the inhibition of essoogenic enzyme

The determination of the hlgltfei.fector, the effect of the M.a.reyes-zihüis..hhibifor becomes immeasurable.

In the case of ACE inhibitory therapy, the triggered bale has inhibition of AGE activity, which may be characterized by the equation.

The effects of a selective inhibitor on the activity of ten ACEs were studied to estimate the required dilution inhibitor, showing a dose-response curve of inhibitory drinking of Captopril ACEs; that;. The stability constant (Ki) of the aditypons fitting to the aditypes by equation E resulted in a brown ACE-eaptoprd Kentpiex of 1.4 J nM.

Other important fskterns are considered words, concentrations of enzymes (AGE) and inhibitors (e.g., nap-cri). The concentration of inhibitor is influenced by the level of the AGE inhibitor drug being used, the drug inter-drug interaction, vslamin, and many other reasons. All these reasons underline the necessity of measuring efficiency. The concentration of the inhibitor is often limited in the maximum concentration of tequinicture, since the amount of available pills is limited and the potential side effects are increased. Therefore, it is expected that the masculine concentrate concentration does not exceed that required for maximal AGE inhibition. Illii) is in the nhi range.

Homogenous plasma AGE concentration was directly determined in 110 volunteers. The carbonaceous AGE concentration was found to be 155 µg agcu (0 Good nM).

In the present invention, it is possible to model the watering of dilutions in the equations of the invention, whereas in this example, the equation L is used to determine the effect of the dilution on AGh activity, the value of inhibitory concentration is taken into account mtg _: standing value concentration “··· nM; Ki ~ 14 J xsM, The calculated ACE activity values (% of active enzyme (E) expressed as total enzyme digestion (Ε + Εί)) are plotted versus high factor (concentration of solution components (£; EE l). ) at different starting concentrations: inhibitor concentrations, covering the entire lysozoic range (1), 1 - iOSo blind)) (Figs.

I is aware that the 12.8-5 IG dilution range is the range where AGE inhibitory (inhibitor-bound) horseradish will be negligible compared to free (dissociated, maximal activity) ACE at the expected concentrations of enptopyril

Thus, it is preferable to select a high dilution factor for the sample where the bound horseradish of the enzyme inhibitor is negligible, in this case at least 2 to 5-fold, preferably 3 to 5-fold or 350-fold, or more preferably at least 5-fold.

In the case of a nymph, preferably the low shuttle factor, where the active form of the enzyme is considered negligible, is up to 20 or 10 times, or very preferably up to 5 or 4 times.

109 902-Í42ASG

The ratio of dilution lactose is desirably at least-14 or preferably at least S0 or at least 7 ° or at least 100.

Important xnegiegyezsk is essential for the present invention to eliminate the highest dilution possible so that the barrier latency (maximum}: eftzi® activity can be saponified, in which case the degree of inhibition can be very accurately determined at 1 and 5 × 2 dilutions (1). This theoretical consideration was tested in a climate study where patients were exposed to various ACB inhibitors, and it was confirmed that the recommended dilution ratios were sufficient.

Estimated inhibition 1 and 5 52 ·· using a high pitcher (inhibit% - fcan) Incoming U Inhibition - And 400- using dilution (% inhibition) Theoretical inhibition forever A-hm?) No cnpiopril 0 0 0 0.1 who is eapiopril 0.7 0.2 2.7 1 µM eapiopril 6.3 1.6 8.2 10 nM captoprii 40.2 5.4.3 41.5 iOO rsM eapíopril $ 6.9 62.2 87.4 i kill captopril 98.3 93.5

3.1, bn Vtó iklo ik «· ^ ethvauk <lm„, tt crv-e

The degree of inhibition is calculated as in the series described in Figure 2.

ACE activity should be measured in a puff up to 512-fold _: dilution, Development of a method for measuring an eosam activity. ne® is absolutely simple. Especially since accuracy may be proportional to the cost of determining. The most difficult part of the invention is the need to measure activity under the conditions in which the inhibitor is present. A number of methods have been tested to make the present invention? Finally, an originally Shmonetla R>;(<sotb, ju »Oh n no < ⁱ tö'm) ~ 0 _n | oo 'o <o _s <« _x , _t) j _{i; we use the totometric method.

A detailed description of the method can be found in the Method description section (ACE activity measurement).

This method is capable of accurately predicting the activity of the hay enzyme in the range of 4 to 40,000 fold. Although the enzyme activities you count are probably underestimated at 4- and 4-fold dilutions, the values obtained may still be useful in determining the effectiveness of the treatment.

Patient code AGF activity .Activity rate 1%) Inhibition (%) 4 • Dilution 400-line dilution whom calculated It measures calculated The 02 1.4: 6 5.83 0.37 147.3 4.0 96 In the 03 2:14 8:57 0.51 202.41 4.2 95.8 A 04 5.55 4.61 0.52 208.54 2.2 97.8

109902-1423

A05 4.64 18.56 0.37 149.61 12.4 87.6 Λ06 1 «9 7.98: 0.38 153.64 5.2 94.8 The OS 7 02 0.47 139.14 4.2 95.8 In Figure 10 0.65 2.62 0.3 120.71 ··> n 97.8 All 0.66 2.66 0.5 AA.27 ΐ í 98.7 A14 156 6.22 0.35 A128 4.4 95.6 ...........DIG............. ............ 1.35 ...... 5.38 0.32 127 Ά 4.2 95.8 A = 8 0.45 ISI 0.42 = 67.66 ............. p: ............. 98.9 The 20th 2.53 10.33 ^:: ......... 0.21 ............ 35.23 12.1 87.9 The 21st JA ..........- .......... 4.59 0:26 102.05 1 5 95.5 The 25th u.42 1.7 ^:: 0:32 128.1 t ' 98.7 The 27th 1.1 4.89 0.44 174.45 ? X 97.2 Λ 29th 0.76 3:05 0.33 13127 2.3 97.7 The 30th 17 4.44 0.6 239.22 19 98.1 A32 0.56 2 ' 1X35 140.06 16 98.4 a33 063 > 2 0:41 162.98 4.0 96.0 In Figure 34 oas 1 ^ 3 049 75.45 2.0 98.0 A35 8.Γ 32.67 0.39 156.49 20.9 79.8 Λ 3 2.4 9.62 0.36 144.62 6.7 93.3 A38 0.6? 2.62 0.32 129.55 2.0 98.0 39 1 = 5 • 16 5 0.39 157.53 2.9 97.1 • \ 44 0.65 2.6 i 0.18 70.97 3.7 96.7 A 4 7 3:28 13:33 0:35 • 4109 9.3 90th 50 9:45 37.82 0.37 = 46.56 25.8 "AJ 51 0:59 2.35 0.38 = 5174 15 98.5 A52 0.63 2A i 0.4 159.04 1.6 93.4 53 0.5 198 0.4.3 173.97 u 98.9 A55 116 4.62 0.41 = 63.83 2.8 97.2 Λ56 = 0.53 42.1 .......... 0.47 187.48 22.5 77.5 A 6 = 4.36 17:45 0.32 _: 129.46 13.5 .......... ........... 36.5: Λ 65 0.53 2.11 0:32 126.19 ........... ............. 17 98.3 66 3.68 14.74 0.36 ..........: .......... 144.26 10.2 89.8 Λ 6? 0.90 3.97 1X39 154.32 2.6 97.4 60 3 11.99 0.38 152.39 7.9 92.1 76 6:57 26,27 0.41 165.44 15.9 843 71 1.56 6.23 0.28 115.87 5.5 94.5 72 9.63 38.52 0:24 95.92 40.2 59.8 73 0.25 102 0.13 50.87 2.0 98.0

FIGURE 2: One mmOl of the azemn ΑΧΈ-inhibition slAtly defined

W99Ö2S 423 / SG

519 patients were enrolled in the study, Mrnden patient was taking some type of antihypertensive drug: taken, approximately xxiiihk: 33 patients received ACE inhibitors. Experimentally determined data from the first 58 patients were presented in the. Table 2 -Sheets, Sea activity values were multiplied by the?, Alkahnasoit dilution factor (4 slices, 4Cfe t to calculate the daily activity of inhibitors) {4-sasres dilution where ACE snh »b vn (t as well as free rhizome (E) vsa) and co-inhibitors (λμλ csSkkeatetil (4-8-fold dilution, where the ACEAnhlator complex (E1) is negligible in concentration relative to s / h3 enzyme (E)). values of inhibition were estimated

Myocardial ACE activity was determined at 4 ·· and Wbsser dilution in patients with hypertension, • The raw measurement results were multiplied by the dilution curves used (4 or 401)) to obtain values for cardiac ACE activity. These activity values are derived from: the golden number of ACE inhibition can be estimated using a single sample,

Transition Filters ^ ons ^ Branches of the Sector hSgihtEfeapg ^

For the purposes of this study, we used the values obtained from patients not taking ACE inhibitors. It should be noted that the present invention provides a surprisingly effective (robust) method for determining efficacy in untreated minlix. For example, in patients not taking ACE inhibitors, we should measure the differences between grandmother's in terms of activities measured at 4-fold and 400-fold dilutions (calculated barrier effect 0 ± measurement error). In contrast, patients treated with ACE gel may need to measure less .ACE activity at 4-fold dilution than 4-fold dilution. Surprisingly, the control group had an inhibition of ΊΜ7% (mean * SD), much higher than the mean expected around 0 (Figure 4).

According to the invention, it is present in the presence of an endogenous ACE inhibitor in brown serum. This further enhances the usefulness of this method for the indulgence of the yeoman, since this major inhibition of eodegem has been revealed to be the cornerstone of the successful introduction of AC in recent decades, despite the intense research and treatment of today's fadiovascular disease. However, our data suggest that serum ACE activity was significantly inhibited under conditions of Zoology (degree of inhibition 723 * 9.6 MA).

We also showed. unlike ACE: expression, the ACE genotype has little effect on the ACE active abdomen. This is evidenced by the presence of an endogenous inhibitor in powdered differences in ACE levels.

We have attempted to identify a putative endogenous inhibitor. Samples were obtained on the sternum using different pore size ultrafiltrates: (Fig. 12 :,), There was no effect on filtering with a 51) kDa pore size filter. In contrast, the use of a 10Å cavity: goreust reflux filter significantly increased the ACE activity of the serine at 4-32-fold hygiene compared to the control. In an experiment using a patient sample, gz ACE-i was double-linked to its associated proteins, and n cross-linked products, human word aibumin (vafemini (ACE) was not shown). The effects of HSA on ACE activity were examined in the following (Figure 13). 'HSA inhibited recombinant ACE activity with 9.6 mg / ml fetal maximal cabacacenifera. It is important to note that the maximal inhibitory effect was 95% (partial gatloser)

189902-1423'SG rog / ml 1'ISA Tissue Transfer Type (Figure 13A). HSA Stripping Stem ». ACE activity was also inhibited. Figures 13B to 6B have 6.1 ICs. Tsmxtal gelation was 95% at 64 µM. At the end, recombinant ACE was cleaved (Fig. I / 3C). Increased by dilution. sxögen ACE activity as seen in the case of endogenous ACE <11/8. Fig.

Therefore, if the HSA-induced gusts are affected by the tilmate series bec-mlpk, the dilution factors will be adjusted by the following series.

For one's sake, "nngas diluted!" wherein the inhibitor-bound backbone is already eluted, at least 5. preferably 8 or 10. very preferably 12, 16, 20.

In the case of a sample, the low dilution factor is where the active yarn is negligible, the juice is 3 or 2.5, and most preferably not more than 2.

The ratio of dilution factors is thus at least 3, preferably at least 4, or at least 5, or at least 8.

There are references in the literature to the existence of an endogenous inhibitor, but this has not yet been identified. Kramer et al. Have described an alteration in the ACE gene: the intracellular membrane region of the protein, which has resulted in a dramatic increase in circulating ACE to 15-fold (Kramers, C, et al., Cottottittos 2001,184 / 1236-48). mutation in at least 8 families, but a. does not cause hypertension or other clinical changes in family members. Perhaps the HSA protects them from the possible consequences of extremely high ACE levels. Eesietrtviteb and co-workers found another ptmtntool that causes a 13-fold increase in serum ACE, but failed to associate it with any disease. (Nesferovítch AB et al. EoS One 2809: 4x8282.). There are some other mutations that cause an increase in ACE levels, but in these cases there is no ibfdöhak cttrdfevascular disease (Semmfer A, et al. Clin Chent bah M'ed 280o; 44'.1088-9 .: Lixtaehank M, et < b. Hetrrolegy 2083/6: 1: 1819-28; Eyrles M el Iodod Chem 2881; 276: 5,525-32,1;

The enzyme serum angiotomyro-stone variase has been pathologically elevated in some graalomatous diseases (eg sarcoidosis, vs. Vascularis. It is also known to be linked to the Ganeher-Fcormin (Táebernün · 1 ski ah N {* !!.! EJ i ^ir 'C'4) rtudds 8 <1 5 ^ 0 ^ 00-4 ^ 5 ^ - ^ %% _A <,% xt \ .t></ k *

In spite of ACE expression, the incidence of arthritis disease is not different from healthy populations.

In addition to circulating ACE, the word aroma aibaroitr also affects the membrane-binding backbone. expected. This was underpinned by Danser and vrm Dijk's trasr. who had ten tendin carriers of angioedema I conversion in different genotypes of upper limbs in humans {Danser AH et al. J llypertests 1999; i 7. IS67-72 .:; van Dijk MA, et al. J Cardlovasc Pharmacol 2008: 35: 484-90.). In these studies, the rate of angiotetri-5 I-0 kx inversion was found in the CD genotype, which is to say that the angtodariosis ratio was similar in patients with H, 1D and DD genotypes.

Previously, several workgroups have suggested the possibility of an endogenous ACE inhibitor, but its nature is not clear, routine by Ltebermaa: et al; Suggested Dilution & Serum ACE Active'm before we measure the effect of endogenous inhibitor. They have found that the inhibitor has a natural frhe and its moiety is 59 kDa equivalent to lberbernvan 1 and Sastre A. Chert 1930: 91): 869-75.).

Our results provide an opportunity for drug companies to develop tip-to-tip ACE inhibitors. These next-generation drugs would stabilize the relationship between ACE and HSA, thereby preventing the patient from having to take daily asgismans 1 conversions.

109982-1423 / SÖ aCE inhibitor. this could improve patient collaboration during treatment,

AllüÜkÉMh ^

C \ CE ~ inhibition drugs can increase your sleep. inhibitors of serum ACE inhibition stallions .ACL-gatlot »zodo patients. estimated ACF. inhibition rate of 93 ± 8%. in accordance with the present invention. <4 abotl Fz r significant difference; (72¾ patients without ACE inhibition, 11 patients with 93% ACE inhibition · s / eao Nvgek e.-eten'í taste suggest that the efficacy of A € E ~ inhibition therapy may be affected even in the presence of an endogenous inhibitor. , individual values are plotted on a histograone, the two groups (patients taking and not taking ACk inhibitors) were surprised: they were sharply separated (Figure 5) .Figure 5 is a graph showing the histogram of an effective ACE- The thresholds for inhibitory therapy indicate a selected line, raised at 9%, if this is a vessel, may be fungal and possibly lower or higher in some '.m / ale. \ kd, this difference would only affect some patients in the transition zone. Patients esy <rseinüea were divided into two large groups, one containing those with sufficient therapy, the mothers with those not.

The hlogram shown in FIG. 5 shows unequivocally the endogenous inhibition of bopv, -z ACE .Ax * (s) patients (rs ===: i58)! its level is in the range of 46- <8'7%. In contrast, most patients taking s-ACE = inhibitors have higher levels of inhibition> - = 383>. et t t'ockzu d beesabetjüa t zt zi <no z t \. nczm / xst is clinically significant at mkmtclt at the ACE inhibition limit (Figure 5).

Connected to scnv.o! sedge

You determine the ACE inhibition of their stones by the moxkmi healing method. we compared blood pressure (needle, Fig.). The; Along with the values of ÁCS activity for 4- and Tax-diluted dilutions, the blood pressure of the patients was also measured. The extent of apparent tax inhibition is given by the ratio of the values measured at 400 and 4-fold dilutions. A. 90% ACE inhibition! value; under! (0 - == 68) and findings (n-318) of patients with arthritic medullary artery were evaluated mat, live in the figure. The bars represent mean and standard error values, the stars symbolize significant differences between the two groups (p-Q.003),

Generated by the monitor blood pressure lowering effect of ACE gáöök accordance awaited kiírókat tests, such as European (Fox _OI: et al Chain Of 2003; 362: 782-84 test what kigttsro's arterial blood pressure decrease .5,8 deíekiálátök, while the EliRÖRA study showed a 6.5 Flgmunes decrease It is worth noting that the EUBÖEA study compared the efficacy of the ACE supplement to that of a single market, whereas our groups were based on the biochemical efficacy of the Inhibitor. similar values demonstrate that the present invention can accurately screen patients for whom ACE inhibition is ineffective.

Activation of ACE under pathophysiological conditions may also lead to hypertension, diabetes, heart failure and other diseases. To date, no significant role for endogenous ACE-hosts on hay has been attributed to the progression of Torgioenzyme converting enzyme-related diseases. Our miadazonfish data also suggest that alterations in endogenous inhibition may be implicated in the progression of disease in ACE media. In addition, our results also suggest that various agents may induce ACE activity by modifying the interaction between the .ACE and the endogenous inhibitor, thus rendering the bowel effect a therapeutic target in many diseases.

ÍÖ9 2% H23 / SG

Sernéiviy.ztrotÍ started

Determining the Inactivity of Inhibition in Deep-tailed Therapy Also, or even optimizing for therapy, is a diminution of the onset of cellular effects that occur. The present formulation is also suitable for providing satisfactory or optimum drug dosage in order to avoid side effects. For this purpose, the inhibitory effect is to be determined, or the dosage should be adjusted based on the values determined. .In the first case, clinical studies should determine the level of inhibition where tnmdntelis is clinical fish. The dose of the drug may be reduced in patients with suppressive inhibition higher than the maximal effective gas retardation delay determinants; They ate. Alternatively, the dosage may be reduced to a value where the patient has no side effects, and today, by determining the degree of inhibitory effect, the patient can determine whether the tolerated dose is sufficient to inhibit the enzyme. treatment of a patient. On the other hand, if the enzyme inhibition is a satisfactory low dose, in this case the treatment of the patient has been optimized. In general, the method described in the present invention is suitable for a lupus dose of an enzyme inhibitor. if the upper and lower levels of the optimum inhibitory effect are available in a circular or predetermined range, and the patient's inhibitory value is determined by this method. Against this background, the dosage of the patient's drug should be reduced if the patient is inhibited! values are higher than the upper hemiertck, or my female bitches if the patient's value has not fallen, i / ak-o limits Optimally, the upper limit of inhibition may not match the color of the maximal effective inhibition, and the lower value close to where inhibition is ineffective ..

The dilution method is applicable to gatohuts n egbusmo-as. when the activity of the endogenous enzyme (low activity enzyme or the enzyme is not. amine n> ^x n \ n nmUbsa eg H'Mö CsA reductase: etc.) or the detesek sensitivity is not. it is sufficient to determine the activity of the enzyme even with large dilution readers.

The key to successful measurement at an angle is the addition of an enzyme to a sample, inaid the measurement of a change in endogenous enzyme activity. In the case of an inhibitor vacuole, the activity of the enzyme is inhibited at low dilution, whereas uninhibited skeleton at high refractive index can be determined as described for endogenous ACE.

This method was tested to aid exogenous ACE Exogenous ACE was added to the patient's serum in an amount such that the exogenous enzyme activity was at least one hundred times that of the endogenous enzyme, thereby measuring the ACE1 activity. A patient receiving this ACE inhibitor was given exogenous ACE for colorectal disease, and is currently being treated for serozygosity and treated with? ACfc activity (Dec 7) The exogenous ACE activity of this serum is shown in Figure 7 as a dilution of the serum of a patient receiving an ACE inhibitor containing expgenic ACE.

The driven result is weighted by equation 2. evaluated: enzyme activity at 0, Sh AEEml in vial and 1: 02.4 ACAnl} at 28-fold dilution. This indicates that the inhibitor concentration was sufficient. to improve his ancestral ACE ak.-u.amt -t-dA-han

I would appreciate the following: to detect the target enzyme, the target enzyme is a bamuben okutEd. Thus, the method can be widely used, such as sm, to disperse the concentration of a drug affecting an enzyme of the Evzoonn nervous system, or

109W-1.423í $ Ö rere5? Rosp; nah, s in case of foie u- A tniaimam az js Ekein: izhnfo. hvgs gv masters bi-brain microbiology; in the production of the esthetic scripts, or in the appreciation of the gadgetrate of natural waste, In fact, Jet technology can also be used to determine the reversible »\» · ·. \\>: νω aS a, ke- í 'toiog i büket! a> \ <ex kram <t i \ u <\ 'u \ o i \ j »a .uc \ 4)

ACE Afi-Focus Measurement Check

Circulating ACE numbness is genetically determined, Aaoteak in patients who are nomozygotic in 56% of ACE gene D afferents! in higher concentrations: found in the serum ACE mimic in those patients. homozygous for the t allele ({> 101 * 7 sg / ml, n ™ 28: ID ~ 115 * 5 ng / ml n ~ 7000; DD ~ IS &gt; ng / ml serum, δ = 56). (Figure lík). ACE activity was also elevated in D0 homozygous patients »although the degree of fallout was 4-fold: blgitivity was determined to be lower (ifo 32 * 2 12 / nd, n ~ 28; lD ~ 31.il tkmí, n ~ 76; DD« 44 * 1 ii / tnl seren »,. S ~ 56), as expected on the machine proverb tet-'vob».

This contradiction can also be explained by methodological errors. To prove the correctness of the ACE actuation, we have linked the algorithms i .ARC to cleaving the textual definition) and the: Anglotenztn 1 conversion (HEEC basic definition). The AUCGG-based assay for ACE activity was found to be proportional to the conversion of angiotension 1 ~ here (Fig. 11A). However, EAPGG cleavage was 20 times faster and easier to measure.

Another important factor is that the hrearnram k -u method did not affect AGE activity when purified tissue ACE was used to perform the ks-tert) ama! 1 z / e · dilution-dependent activity of szMnm.ACE (Fig. VB), when four biphasic females were exposed after an initial increase in activity (to diluent dilution) to a plateau of sulfur A «Io-M fold ingce followed by a further rise (64 fold dilution down), (Figure 1B) These are adamk ccv for the presence of endogenous tninfmer. In the case of diluted serum, the inhibitor binds to ACF, and lives on the akin dosah (barrier); rahhuro Ivraokítz \ G1 -re-l, foheatí. its file.S, ote (ti (figure)

Dare · 1 Y understood

Blood samples were collected from volunteers using standard aseptic technique. The blood now nahv) cartilage were incubated at room temperature ,: serum fraction was separated by 25 {perrag ISOŐgm centrifuge) and -25 ^c C tsrektík further use. Genital Balse ObS was isolated from Andkösgulah venous blood using a DNA isolation knife (Etexigene DMA kit, Cak No, 51204; Qiageu GrabB).

ACE akíivitáu 37 ^c C was determined by means of an artificial szubszírái (N- (3-E-t2iuryijacrajöyl'j pbenylalanylgiycylglycine; Sigma-Aldrich,.. No Cai, M 131) was reakcíésiegy 25mM Hefest (Nr2 -hidxtehy1?> tperazIHe-N-2-ewwolfonjc · acldi, (1.5 mM F »AKX3, 30 * 3 tnAf MaCl, and serum was torotroa / ou apBh.2). A mora 96-well plate (Cremer -Slo ancestor; Gat, No, 655101), with a cell reader fNovoStar platform read BMO Labtech GmbEl), Absorbance (341) sm) change (340 rtnil every 5 minutes, 00 rpm). ACE activity is as follows:): numerical values · Acute cascade - (<h) * D where 8; slope of the decrease, kr optic skate reduction when t nmei FikEö Ixnaiik, ö: ACE activity is expressed in units, 1 1.5 corresponds to 1 so-called dilution factor tot

1 (10402-142 ESG: 9

FAPGG Degradation In a few snappers, the reael mix has also been tainted in a brown hundred albums (HSA Cat. No. :: N1-201908-21 1, Hornna SktPtena. Mhaa & síanfcg: and tradtng ΓΗ)}.

lake A £ A etotoFto5aSS: ÓAiA% Aíl

The UAE rnFerrucleate lobe genome was determined using a standard protocol based on the RI and D allele PCR based on the Si-sphScope (Riga B, etak J Óin shot 1990 ^ 6: 1343-6.1, product number 59). pollen was isolated by gel electrophoresis and visualized by etheton-ferromide staining The presence of alleles 1 and D was a product of 490 and 1:90 base pairs respectively.

A sample of serum from a healthy volunteer was ultrafiltered with 250 and dilution of 25 and 10 <RTIgt; tour the volume of k \ mduiass \ n> í.zUomensueü ukíAj pre-energize k-mduiass.

! ·> Aogfeáíszislko ^^

On the sternum: 37% of the samples were 1 strain of gut 9, -5 pBi of Anglo-isoenzyme 1 (synthesized by GeaScnpt USA Ine.), 30S: oM (i to nA! t .tum, e'egther .g.> g \ .ea / noA reaction .. The: a-tgiotensin pile fillet was measured after the Quarter was stabbed with a horse's mousetrap (Vivaspta .500, Cat. No ,: VSO10; Sartories Siediot Sloieeh), Siumsdzu HPLC and rewz fission ClA column (Hypersd Goid, Cat. No: 25085-) 54630- Thermo Scfeníiftci. TFA, Eigms-AldrieN CaL.332831). in water up to the eluent "B" contains 0% TFA4 in aetostotrilylstrom-Aldrich, Ca. N;>. 3499b}, The: pggids are separated from one another by 22% by 55% aestomtotoe ovo gradient: The Anglofenzyme peplides: with a diode-array detector up to a wavelength of 230 m, the pepids are present. (peptide) calibration curves. The amount of sngmteuzin peptides is plotted and aligned with the reaction time. You give the rmgsoierszin I conversion formula old: 1 omol asgiotensirs I cleavage by Imi serum during l

1-6 Defining ACE.me.a! Ity. The amount of ACE in the station is one. commercially available: commercially available AGE El .ISA developer kit (S. & F3 Systems, Cat. No.DY929) deviated slightly from manufacturer's instructions. Briefly, the ELISA plate on $ 0 agar) was coated with capture antibody, and the remainder of the binding medium was blocked with reagent-diluting solution (Galbeole's half-shot / mg bovine serum pbmninin diluted in blood). , and then the Kotlée of the antibody-amygéo kcwvfewkef was marked with a desiccant antitostere (20 ttg / pocket). The rvroxtdu / t component of streptavidbi kcmjugáh horseradish diluted 200-fold) was added to the pockets. Finally, with a solution of sucrose, t (i, 3 mg / ml ternunedt-bettzldto oil μ.; H; O> 50 <; M V'vtu daruin ka aompiev moss, u <, g <.tsi v.kemt \>, νι, The optical density of the samples of η. nl, 5 it1 »> i'.Yc'tuk te \ was measured at 450. The samples were measured at least three times or at least three times the standard deviation below 15%.

1N Crash Cover

The AC & AHSA. coupling effect with tofembsfonketonal cross-linking agents: staClyized. Starting point: Grace was watered for 1 hour at 25 ° C, which was diluted 1.25 in diluted serum, 25 nsM K £PES.

109002-1423 / SÖ

VmM (+}, or 2.5 tsM., Or 5 ntM ¢ + -: - 5-) strcctnlnidid-HN-mateissopioamidrOrótxfsefisén <. xi is' {.t t + Mtl ' ¹ vt D \.>>>. »ukv vet Ni ^ 311'i k' - '' m 3Í [\ -nmc lm.>m> n _{> x} it» nd <> bes: a: Ethyl glycol - north RSM (FEGv, Thermo SclentiEc, Gat, No.221t) Sh 7,2-pti-a iartaine wheat Exclude non-cross mrslekuláka 56 Λ trifihydroxhuetil-ınirtontein & Ínirtontei Cat. N0.T15O3) Stimulated for 3 minutes with SXObacea, Biotinated Human ACE Goat Antibody (R&D Systems Itse, Patt No.84366O, 22.6ag) and scintobilized streptuvidin resin) Fterce, Gat Neok. in order to bind the Kefesíkoot Products to the surface of the resin. Blind 22.6 ng / ml of non-specific goat contained IgG instead of ACE specific antibody. After incubation overnight at room temperature, the resin was washed S-times with 25 mM HEFES, pH 7.2. A. Purified proteins were eluted from the resin by stitching for 10 min in 2x SDS in shuffle buffer (Tgglma-Aldrteh, Cet No, S3401). The ligated proteins were visualized by 5- 15% gradient gel electrophoresis (Mnfi-Pro) using Western blotting analysis. Purpose, Gat 00,450-1086, Sio-Rad Laherstories Inc.), Primary antibody 1: 1000 diluted: Goat antl-ACE antibody (Patt. No, 841365; FfeD Systems Inc.), secondary 1 :: 40000 inghasd peroxidaz: coajagans were an anti-goat antibody. The biotope was developed on a green-sensitive x-ray film (Frimax Berlin GmbH, Gat. No, 7) using Waste .1 .. Igbymng P1 us- £ CE reagent: (Eerkln Elmér Irfe Sciences, Cat, No .Net 10400).

1-7-1 Nlp ^ üngggruntés. ^ Pathological HEAfiergS

Previous experiments: Your whole serum space with FISA and 100 kfe pomsutal space was also done with ukraine filtered serum. The initial: reaction serum diluted in SD-diluted serum, 0 mg / l (+), or 24 mg or 48 mg / ml (-5-5-5-) SSA and 5 IhAl SMliFEGfi contains 25 mM HEP.ES buffer. 2 ipli-n. The following steps were the same as those described in Rorabta. The Western blot after recall from the membrane during the antibodies eiíávr-lhrsttnlc 20 ^of rim 60 C ..stripíng "puEerbeu which nátrinm-dodseOsznlIáfet {% <SD'S · Serva Gd \ k- ,, .VTCí 20 nim DI -d nonenol (Difi, Emma \ ldrcb. ("í \ e £ h ^, '' N)). o« udfi [ _n , in purfer, 7.5 pli-n. The development protocol was repeated bfetináh human serum albmnln diluted 1:50 -806 in gold primary antibody: (Exbfe Frába, Cat Nu..fB-257-CIC0) and ICA® 000 diluted with sympathidine peroxidase (asexon, Cat. Ne.0 6-030-084). , mount perexidází connect to the complex.

1, .7.2 .Recotnhhdm ^

Recombinant ACE-HSA interaction was assayed on EL1SA plate surface (Gremer Bio-One; Cat. NO.05506I). 300 μί of Fefeus HSA dissolved in 300 µl of tight powder (FBO pi 7.2, content: KGI 2.7 mM, ORFF → 1.5 µM, NaGI 137 µM, NafiFG _if 8 µM) was added and punctured overnight. at room temperature, mount the HSA to the surface of the plate. After washing with EBS, 100 µl of 625 pM SMFEGJc and SMP G G: _i2 were added to the pockets and the plate was rinsed for 30 minutes. After a thorough wash, 100 µl of 260 ng recombinant ACE was added to the pockets. ® Sysiems, Fart Nö, 84l3é7) for 1 hour. 100 µl of biofilm human goat anti-ACE antibody (F&D Systeiss Inc. Part bio.84l36h, 22, she jump) was added to the plate for 2 hours after washing. Following washing, 100 µl of strepiavidin-1 conjugated tons of puroxide (R&D Systems. Part N0, $ 0O2 SALT3) was added for 30 minutes to the wltek; Finally the moored. The ACE-defective anlhest-streptavidm IIRP complex was visualized on a sucrate cclao &lt; 1 &gt; (containing 80 tnM acetic acid, 0.4 mg / ml of tr.tetracetyl bertzidi, 0, and 1 µM l'EO;) using a 380-ohm order. : crossbreeding agent was treated with ΙΟΟμΙ üfihnM'-osTris-HCl solution (2, estop), or extinguished: a: bulb (column 3), Anc-m specific binding 1% ~ • m zsclat'u solution ζ5 ev!>' p \\ y \ ik, -,.; tkom b nsknksvs nCkui 11 o \ Jop> tes / telm ». as a control for fesus, a sandwich ELISA kit is available from the EtJS.A kp Helpdesk

100902-1423; SG 'λ!

In the other case, triplicatexnoxics were performed.

S RdSABbíntogACT

Recombmans ACE: was prepared using the Ssc-te-Bas Topo expression system for Lavirege®, Cat. Ne. Al 1101}: according to the manufacturer's instructions, RrMas: competent for the purgeniia. £ <: <> & · strain (lavitmgea, Gat. 10 No.G8620-33) with pin gene containing ACE gene: transEirntals: (eneeeneCopoeia;. Cat. No. EX - T7349-9191), Following construction of the pEasISae containing the ACE gene, pEasíSae is competent in DH1 OBac. coll ((nvitrogem Cat. Ne. 18361-012} was stained for preparation of reEaotbtaant bactnid.), The bactnid DNA is SE-9 insect repeke (invirogea, Cat No.: 0362-100} article / föktahnk. Further SE-9 cells were infected with this baknlov meat, and on day 4, the insect cells were subjected to fee-tritrifugation (10,000 g, 1b min, 4 ° C), and the pellet was washed in PBS to remove the sciatic digestive medium. RIEA .filled (50 ssM Eris, 150: mM: NaCk f * Triton A 100. 0.1% SDS, N deoxylated fatty acid was homogenized with the aid of an identifier. The supernatant was added to O 500 g, 10 rt, 4 ° C and on an aliquot (Rnauer, Biotbs 40 psn Q anion exclumge coltunn, cat. no. Y4 (5) 1) 25 mM HEPES, 15 mM NaCl was injected at pH = 8.2 in a stock solution. AzACE was eluted with a gradient of increasing concentrations of NeCi solution (vectors at 540 mM-mg nC of collected Iraklocks also measured the ACE peak by ACE activity).

The assay was performed with serum (in various dilutions®),,, mM mM substrate. (PAPELG. H- (3- (2Euryl} acryloyl) -1-phenylaminophenylglycyl, glycol, 3,000 mM Na 2 O and 25 mM HEPES) (442- (Eds. Xylan} uli) erazine-1-ethanesulfalic acid ) pH ^: 8.2 solution®, λ measurements? '56-ivukj wBelon filters _{were made with} Gien "r Slö-OneG 200: or 300 μΙ-ss- final volumeibs®. A37" G ontlka densification 340 nm of the NovoSaar plate render was adjusted by linear alignment (values are eluted if the fitting is r> 0.9). Calculate ACE activity as follows (7, equals):

Activated-solid.HNb, 133} ^ 1993,742 * 0 (7) where and the slope of the decrease of the optical board, ODO is the initial optical wavelength, 0.133 is the dorsal surface deuser (25 mM HEPES and 30S: mM NaCl), and 1093,792 OO change accompanying substrate completeness. Bt alkaline bowl,

Baseline characteristics were compared with the ebi-squared test or Fishez's exact test (qualitative properties), while the Ssnden's t test or Msna-Whitnev li test was used to evaluate the quantity of swans. Statistical package SPSS 15.0 was used for iSPSS 35 Inc., Chicago, II. ESAs, and a p <O.OS significance limit was used.

TERMÉKFEJEESZFÉS

An apparatus according to the invention

The metering meter of the present invention comprises at least two minefab gadgets capable of accommodating a sample, each of which may contain a plurality of specimens at the same time. It is designed to receive mimics in different volumes.

The meter contains a dilution unit which can dispense reagents to the sample container. Blending after bleaching provided by the equipment var ·. This can also be accomplished by the shape of the dispenser which expands. If possible, the container should be round in shape to avoid mixing points.

Í09902-14237SO

Spice gauge for gauge »g tolerances:

The meter is equipped with a dst brush brush system, which dstectins and is able to follow the reactions of a tome, thus producing the Jaio avt atom. The spectroscopic rettus may be a spectrophotometer system, which may, for example, include an essential and a detector; or a fluorescent detector resstaep or a VV-LF (µV-VS) overlay system, depending on the & reaction component; and / or the products of the reaction or any other method by which the activity of the enzyme is measured. In the detection system: preferably conversions of the enzyme's tailbone; followed by synbstrate or reaction product.

A preferred embodiment is a plate reader combined with one or more mixing units and a foam system for delivering the bigito solution and / or reaction components &lt; / RTI &gt;

In a preferred embodiment, the inhalation test is provided. In this sense, a disc pocket is equivalent to a disc reader for a tracker. In a preferred embodiment, the cuvettes have different volumes, since the ratio of the cuvette's essence to the different volumes is lower, and the ratio of the volumes to the cuvettes. Preferably, whisker vultures are autumn or even uniform, since volumes vary widely.

The meter may also be equipped with a data processing unit or a processing and data storage unit, if possible. These units are operatively connected to the inventive measuring device. The storage unit contains four software that can:

control the dilution unit for appropriate dilutions and / or obtain data from the detector unit and / or perform the necessary calculations according to the method description of the invention.

If desired, the meter will operate fully automatically.

The invention also relates, preferably, to reagents or components selected from enzyme templates, enzymes, inhibitors thereof, diluent buffers, reaction buffer. Preferably, the components are in kit form, preferably together with the device or a suitable part thereof, e.g. a cuvette or a medium that is provided with a program.

In a preferred embodiment, the sample or sample tube juice is labeled, for example, with a sponge, and. the instrument is equipped: a barrel of salt that cooperates with the processing unit. This allows the meter to automatically defen »dilute and inhibit! level.

.fez With regard to the € E ~ barriers, a. I find whitewashed limestone for practical application. This solution: special »piefortife. Sakeinber is able to introduce the method to other targets eg. eazymphohibory pairs in my case. In this case, the method, especially with regard to detection, should be optimized.

Further developments may target the volume of canbovascular patients, for example, and specific; ACE, HkfG-CoA rednectase, rennia and Or dipeptidyl-peptylase-4 servants. .Another area of development may be enzyme-linked monitors used in psychiatric or psychosomatic diseases.

RESULTS ENV EC SUMMARY

The utility of the method described in the present invention has been demonstrated by azACEi therapy. $ 501 in a cohort of 501 patients with high blood pressure? patient's ACE-gsdo is prescribed as therapy. The method was tested in these patients, and 82% of the 387 patients showed adequate inhibition. In patients with insufficient inhibitory activity, we attempted to determine the causes. These patients were contacted and called upon the Physician to consider the importance of ACE inhibitor therapy. They were targeted, and their therapy was found to be ineffective, and they were returned to the LöjwH study, and 45% of the patients were out of control. Increasing the dose of ACEi in patients with pN. leads to results. In the 1770s of the patients a. control test showed satisfactory inhibitory effect, in another 17%. due to side effects: I had to switch to another drug. Patients did not at Ladder's. found: the cause of ineffective ACE inhibition. in these cases, the M who tet.tpt.3 med '\ main,: VtÍzzv.iM?! tuk

Patients' blood pressure showed strong kmrelam with effective inhibition (Fig. 9).

IPARÍFEÍKASZNÁLHATÓSAG

Who lalálmány for Atzmon tmgy educate enzirngásló medication '' _{{\ »u. vi pu. uoo v <ta! Insufficient therapy puts a heavy burden on the social whistle-blowers, thus increasing the significant economic role of personalized treatment. The first step of guilt in estimating the efficacy of ongoing or planned therapy, Leien's invention can make a significant contribution to this step. by providing a simple, one-step, single-step, single-step method for measuring the efficiency of an enzyme mnibitm pr, particularly for ACEI therapy. Too much salt, the present invention provides the opportunity to adjust and optimize the patient's therapeutic regimen to reduce unnecessary burden on the patient, thereby increasing patient compliance.

In general, the invention is capable of effectively and simply testing for the presence or absence of an inactivator in any sample, since the method does not require any further sample or control. On the other hand, the method described in the present invention can be carried out with reagents such as succinate or a commonly used enzyme. Because of the potential for automation, this method can be carried out without the need for expensive or overly specific equipment.

HÍVAl'KGZÁSGK

1. Pfeítér MA. Braunwald E, Moye LA, et at. Edéét of csptepril on rnortahty and freshhidlty in pattanta wnh leit v <ístté'tthír dvsijmatiou aftcr myocardial Införctfon. Results of the sarvenic enő veníriealar enlargement trial. I eb Ά E ίη \ e ^ tsgatörs. N Esgi JMetS _1992; 327: őá9 ~ 77th

.. Ccn el P, Mu'uaá A, Soubrier E, Wllhstntí TA., Recést advaaees; in knowleáge ot the struviure and fesetmu oí the angmtensm 1 eepverting esysrte, J Hyperífitts Srtppl 1995; IXS3-shoot.

X Hóhér C. TToaot AM> Coryol P, Sonbtier f, Strueture o the angioteosin I-eonverting enzyme gene. drinking aitennate pm morale eorrespojíd te evoláueuary steps of a duplicate gene. J Bioi Chem 1991: 2: 15377-83.

4. Oppong SY, Ifeoper N'Mi Lbaraeienzatios of a Secretase aeilviii refinements: augiotensimeonverting errzyme ften; t'ue. memlmme, Biochem, 1993: 292 (Pt 2); 597-6.

5, Weíi L, Alherto-Gdas F, Soshrser E, Mfehaud A, Cotvol P, Cianset E. Expresston See Characterization of recmrsbinam; human augiotensiml ooavsróng enzyme .. Evidenee fer a C-termm & i tmusmembrane ancbor and tor a. proteolytk '. psoeesstug of the serum enzyme; mnbin; mt and plssma enzymes Biel Chem 1991; 2f> 6; 5540-6

She. i v. \ KM rtíkaey el permdoprd m n'dnetmn of eardwaseol irish evems ameng pattom * ndh sttbie emvnms sríery diseaser razed, doubfc-biisd, placebo-coatrolied :, nmlíeenbe trial (tbc EUROPE 2: 7et; -8.

7, s \ mo 't' H eus <t> ^ri t »e íme r 'ti b- nop' í r <irmMkrn I gLsv. G m «<te v sgh iütnyuk váílidravm six tveeks _; .ate aome ntyocaröku itstarcHoo: the GíSSI-3 tnal. Gruppé judaliano per Here Slndlo ölel

Sepravvíveuza nelflnferio Mioeardico. J Ast Col! Csrdiot 1 996: 23: 337-44.

9. ISIS-4-: a. rsddorsised feefcífel written assessiag: early eagle eaplopríl, eagle monersitrafe, and iatesvösess oagáesiuaa sslphaíe in 58,030 padents sfeb saspeeted: aeute? uivocardial Inlsreden. ÍSÍS-4 (The Great Lord Israadonsl Smdy of Leiarct Survival) Colhihoíabve Oxoup _: , Lanee! 1995: 345: 069-: 80th

9. Cohn JN, Johnson G, Zxesehe S, «5 ab A cooipatison ot easlapríl wöfefeyrirslazmo-Koserbfes dináraié fe ibe treanneni of chxonie congestive heart fadm's. fe Eogl .1 Msd Í99!; 325: 393.

10. Greenbetg B, Qainenes MA, Korlpilkd C, ot al. Effocss of lougrierm? enalapdl feerapy on cardlac feructure and sponotton in tip »wish lob veatricnlar dysftstetton, Réséit» of íhe & GLVD ochosardlography »nbsnjdy .. Ctrenbhon Ι9Ο £« | 253 M

11. tnders 9, Sebraáer J, Beigér 3, et al. The PHAEAÖ ski: prevondoa of hypertesfeot; wtfe tlre angfetonsiíreoavertirsg eazytee inhibitor mndprtl ® partem »wltd higE-norm! blood presssre; a prospsedve, iron skates, eoadolfed prevenilon tria! of fee 1 Hermán Bypertessiort Leaape. .1 Bypesthet 2008; 26: 1487-96,

12: Cospídá C, Maiesaa ML, Valagsssa L, ski al. Comparative Efiberi of Oandesarí sad sad topi is found: veniratoarar hyperireplty $ ® parteat »Wd essead & l Itypertenalon: ihe -candestixtoa messjnextt in tbe creatnatto of caedsae Eypertrophy tCAfClí) 20, 2200: 22.

13. Raggenenh P, Fassi A, fili AP, e al, Frevendng. mJemihiminuíia fa type 2 diabetes ». N bogi 5 Msd 2094: 35 i: 1941-51.

14. Hoogwerí BJ. Youag 3B. The fföPE adidy. Ramipril lomered earworm risk, vitssoin E did Pót, Cleve Clin J Med 2900; 67: 28- <· 3

15. Rigat B, Habert £, Altome-o-eris E, Cambien E, Corvol P, Swbder E. Aa toserdon / deferred polyaiorpdisjn in ihe angtoinensin 1-eonvsrdng idea genes fed by an aerial enzyme suppressor. 3 Cd® Loves 1990: 80: 1343-6,

16. Cambien F, PbJrtor ö, Eecerf L, el: ah Delefem pelymorphism in ide gess for artgloieasloOesvorísng «rtzyme and potom rssk feetor far rnyacardlal mrixcítoa. riafere 1992; 359: 641-4.

17. Sfessaes JA, 'Wbng JQ GrsoecltirsC, et al., The deletion of the polymorphism of the Ide angistensin etisyphilis gese and cisovasovasarnslsk, J Sypsriens 1997; 15: 1579-92.

18. Lsadpsfeíneí · Κ »Ffeffer MA». Kieste R, et al, A prospeedve evaluaöon of an tsagjötonsín-öoawöögr ^^ jróe. gesre polymorpbisai and the risk of hesad disease. Mfrigid Mcd 1995: 332: 706-11.

19. transversal C, Dsrdlov SM; Öeioum f, ski ski. Folsí maiadon And ihe stslk of angioiensfe-converdrtg eozytne estise »a drama bíerease fe sertsn aogietesisls-oeovertlng: enzysne bst no oardievascoísr driease. Cricrtlarien 2901: 104: 1236-49:

20. Mesterovsich AB Bogarth OFF) Ádsrielsev _VA: et sl, i-Angidtonsm converdng eozyme maiadon i'l'rp 1197Stop) estis »the dnsando fecmtse dr blood ACE. EtoS Cfee 2S09; 4: e8283.

21. ^ emmier A, Sióm R \\. triplan I fe.nifev SM, klekgetber I f irstbank M. Heredí'art fetpoi-ACl 'erm.s dnstö fee Pí'ol 1991..eu muiatíouof somatic ACE as a potonoal parii! tn di.toaosrs; a Orsi: iainíly ostslde Enrope. Cbs Chem LabMed 2006; 44; 1088-9.

22. Litmefemk M, Ke »via K,, le« b M. et al. The feed enzymes are ascertained by the enzymes asggesting the nesrosamoid. blenrolpgy 2003: 61? 1819-20.

23. Eyrfes M ?. Mlohaud A, 'Deitium 3, et al. locreased shedding of angrotensla-coaverdng onzyrae bv the literary editors !:?, ihe slatlk region, .1 Btol Chem 2091: 2 ^ 6: 5525-32,

199992-1423 / SC

5: 5

24, Uebennaa .1, Bsatlsr {Üeredíen of ssraaa angtemsiss-coaverthig esxyíse in Gasekefs dísease. Esgl .1 Med $ 76; 2 $ 4: S442-4,

25, Siuddy P, Bírd B, Refer DG Sérts® aagisfeasiu-eoavertíag eazyree (SACE) for sareredösis aaá olher grutrelerestot disarders. Lases 1678: 2433 1-4.

26, fhres & r Ali, Beiarea .1, Österöp AP, Adraréaa! PJ, Sehslekarep MA, A & gteSmm ϊ to asgísíensls: M caftversíoa la tóé breare; -feröan® aad air. Eilect e l lake saatóiensi® esnveroag for eating gsss iaseAlan / delsdöa ptóyreorphAni, .1 Myperteas $ 199; 174867-72.

26. vsa Dók MA Krooa 1, R; nnper AM. Bconasma 1 \ Dasser AH, Csatig PC. The angwler ^ tn-cen'.crtmg esyme gene puiynuirphisni and tesponses io angioteasins a®d hradykiaia is tóé brown® rerearm, j Cardunasc Ptemacoi 2öd0; 35,484-96.

28. Llebensua 3, S & sire A. As axíghjtessia-eouvsríreg esyw i ACfc) rtórbasr 'in hstsaa serre® Iscressed ssssiiivity csf Ibe sernm ACE aasay ibr deiestisg aeíivs sarctódosi.y Cbesí ΐ «*. .

189962-1423 / 861

PATENT CLAIMS

Claims (12)

PATENT CLAIMS Determining the level of reversible inhibition of L asgiotensin converting enzyme (ACE) by which method; (a) obtaining at least one starting sample, (at least one aliquot is lost: from at least one starting sample, (i) preparing reaction mixtures containing: the olefins and, if desired, other cycloids, thereby producing different Hylotics fecal shells), and Thus, it is noted that dilution results in a different ratio of the active and inhibited forms of ACE in these reaction mixtures when an ACE inhibitor is present in the at least one starting sample, (d) measuring the ACE activity in these reactions, and measuring the ACE activity values measured. . (e.s multiplied by the measured ACE activity values multiplied by the rseksk corresponds to the responding Viktors to calculate ACE activity values are obtained, (1) determining the level of reversible ACE inhibition present in at least one of the starting samples by diluting with various dilution factors: a? difference correlation! shows a level of revolutionary inhibition, whereby the dilution factors are determined to be statistically significantly different when the high dilution! lactone; value is compared to the low dilution lacion! value when it is; an inhibitor is present in the reaction mixture The method of claim 1, wherein the ACE is present in said at least one sample, The process of claim 1, wherein the azACE is added to the reaction mixture as an additional ingredient, 4. Demand poultry scatters; a method wherein the at least one starting sample is a biological minimum obtained from or obtained from a biological sample. The method of claim 4, wherein the at least one starting sample is blood or is derived from blood, She. 1-3. The method of any one of claims 1 to 3, wherein the at least one starting sample is produced by the action of microorganisms, 7, 1-3. Claims Bomb Process, wherein the at least one starting must is obtained by a process used for the production or development of i-isblor. 8, 1-7. The method of any one of claims 1 to 3, wherein the method is for determining the efficacy of a drug therapy; carried out 9, T-8. according to any of the demand port; procedure where the dilution! Factors to determine that the ACE activity values you calculate will give you at least a factor of S or greater, more preferably at least a factor of greater or greater. In ACE activity values, when the high dilution factor value is lower than the low dilution factor value, an ACE inhibitor is present in the reaction mixture. The method of claim 9, wherein said freezing factors are predetermined by a predetermined dose-abdominal relationship between the predetermined mhjhfier machinery of the at least one starting sample and the inhibition of ACE with an inhaler, Π, of claim 9 in meshed Eija,, ^s u crniitett no dilution factors are determined experimentally Oton · bed; that we have different dilution factors in one. in the deftmal method of claim 1, 12, deactivation of ACE substrates and optionally additional reagents as shown in Figures 1-11. claims 1Ö9902-H23SÖ? ^{Í 120ö2!} 9 nz ACE. reversible inhibition of said iah ^ chemical to determine levels, IX Isolation of Isolated ACE Ten 1-1 i. according to any of the claims; A method of providing a reversible inhibition of the ACE inhibitor abnis to determine the efficacy of a medicament therapy, wherein _: The use according to claim 13, wherein the isolated A CB is purified or recombinant, 15. The use of reagents different from your thresholds is illustrated in FIGS. Elimination bout according to any one of claims 1 to 6, for rattling ACE activity, horse. Preparations were made for determining the amount of false ACE inhibitor in the enzyme-producing enzyme. Any of the following claims, wherein: - means for receiving or receiving at least two aliqnotes from at least one initial sample, - means for adding additional components to the aliquots; iétrebozására, means for diluting the reaction conditions with various dilutions Shkíorehkak - a means for measuring ACB activity in said reaction mixture to determine the measured activity values, - means for determining the degree of reversible inhibition present in the at least one chimpanzee sample, which is provided with a data processing unit and a storage unit operatively communicating with the measuring apparatus of the invention, and a program for controlling burns comprising: - ehege.vv λ Necessary calculations according to claim 1 for multiplying the measured ACE activity values with an appropriate dilution whiteboard! and the ACE activity values thus calculated do not correlate the difference between the ACE activity values so calculated with the level of reversible inhibition. 17. The method of any one of claims 1 to 4 for determining the degree of reversible inhibition of ACE by an inhibitor comprising: optionally, the device of claim 16. - the ACB extension, - ACE- · isolated horseradish, where appropriate, - an ACE inhibitor in the transient case, optionally, a diluting sulfur.